CN110512018B - PCR primer and kit for screening cabbage self-affinity material and application of PCR primer and kit - Google Patents

PCR primer and kit for screening cabbage self-affinity material and application of PCR primer and kit Download PDF

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Publication number
CN110512018B
CN110512018B CN201910401144.0A CN201910401144A CN110512018B CN 110512018 B CN110512018 B CN 110512018B CN 201910401144 A CN201910401144 A CN 201910401144A CN 110512018 B CN110512018 B CN 110512018B
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cabbage
self
pcr
kit
screening
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CN110512018A (en
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吕红豪
庄木
肖志亮
方智远
杨丽梅
张扬勇
王勇
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Abstract

The invention discloses a special PCR primer combination for assisting in identifying cabbage self-affinity, a kit and application thereof, and relates to a molecular breeding technology. The PCR primer for cabbage self-affinity screening is BoS1F/BoS1R, and the nucleotide sequence is shown in Seq ID No.1 and 2. The primer or the kit provided by the invention is used for assisting in identifying the cabbage self-compatibility and/or assisting in screening the cabbage with the self-compatibility, and the result is matched with the field identification result. The PCR primer, the kit or the method has the advantages of simple and easy operation, strong specificity, good stability, capability of realizing early breeding and the like when being used for breeding, and has great application prospect.

Description

PCR primer and kit for screening cabbage self-affinity material and application of PCR primer and kit
Technical Field
The invention relates to the technical field of biology, in particular to a molecular marker for assisting in identifying cabbage affinity, a special primer and application thereof.
Background
Common head cabbage is abbreviated as cabbage and belongs to Brassica species of Brassicaceae (Cruciferae) Brassica. The cabbage has strong adaptability and stress resistance, and has high nutritional and health-care values, so that the cabbage is widely cultivated in China and even all over the world. In long-term cabbage breeding work, breeding units and seed companies mostly use self-incompatible lines to prepare hybrid seeds, and the key point of many unit breeding is concentrated on the self-incompatible lines, but the breeding of the self-incompatible lines is neglected. The hybrid seed prepared from self-incompatible line has false hybrid phenomenon, so that it can promote the wide utilization of male sterile line. When the male sterile line is used for preparing the cabbage hybrid, the problems of high artificial pollination cost, low seed yield and the like exist in the bud period. In order to meet the requirement of cabbage variety breeding, the maintainer line and the hybrid male parent of the sterile line are preferably self-compatible lines, so that the breeding of the self-compatible lines is urgently needed to be enhanced.
Self-incompatibility/self-compatibility is defined as whether a fertilized egg can normally form after selfing and pollination of a normally fertile hermaphrodite plant in a flowering period. The fertilization process can only proceed normally when the pollen and stigma are foreign pollen of different genetic composition. Cabbage has obvious self-incompatibility, and the research on the utilization of self-incompatible plant hybrids mainly focuses on the self-incompatibility, but the transformation of breeding approaches prompts the wide utilization of male sterile lines, and the people are prompted to pay attention to the self-incompatibility which is a relative character of the self-incompatibility. The subject has hundreds of cabbage self-bred line materials with excellent properties, wherein 87-534 are strong self-compatible lines, which provide materials for cabbage compatibility research and improvement of self-incompatible lines. The field needs to develop a PCR primer and a related kit for auxiliary screening of cabbage self-affinity materials.
Disclosure of Invention
According to the requirements of the fields, the invention provides the PCR marker for cabbage self-affinity screening, which has strong specificity and good stability and can quickly and effectively meet the requirements of the fields. The technical scheme claimed by the invention is as follows:
the PCR primer for screening the self-affinity material of the cabbage (Brassica oleracea L.) has the nucleotide sequence as follows:
upstream primer BoS 1F: 5 '-AAACTTGTATCTGAAGAC-3'
Downstream primer BoS 1R: 5 '-TCTCGTTCTTGCTAGTCTTC-3'
The size of a characteristic band of the cabbage self-compatibility material amplified by the PCR primer is 93bp, and the nucleotide sequence of the characteristic band is shown as Seq ID No. 1.
The kit for cabbage self-affinity screening is characterized by comprising a reagent container containing the PCR primers in liquid or powder.
Also comprises reagent containers containing reagents required for performing PCR and/or electrophoresis.
The method for cabbage self-affinity screening is characterized by comprising the following steps of:
taking a cabbage sample to be detected as a material, and extracting genome DNA of the sample;
the genomic DNA obtained in the step is used as a template, and the PCR primer is adopted for carrying out PCR reaction to obtain a PCR product;
the PCR product in the step of detecting by electrophoresis,
if the PCR product of BoS1F/R shows a characteristic band of 93bp, the sample has self-affinity characteristics; can be used as a self-compatible candidate material;
if the PCR product of BoS1F/R shows a band of 80bp, the sample has no self-affinity characteristics and cannot be used as a self-affinity candidate material.
The PCR reaction system comprises: the total volume of PCR reaction was 10. mu.L, wherein the upstream and downstream primers (10. mu. mol/L) were each 0.5. mu.L, the Whole gold organism 2X EasyTaqPCRSuperMix 5. mu.L, the template DNA (20 ng/. mu.L) 2. mu.L, ddH2O 2μL。
The conditions of the PCR reaction are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 45s, 35 cycles; extending for 7min at 72 ℃; storing at 4 ℃.
The preparation method of the kit is characterized by comprising the following steps:
and filling the PCR primers of liquid or powder into a packaging box marked with the cabbage self-compatibility screening application.
The preparation method also comprises the following steps: in the package, reagents required for performing PCR and/or electrophoresis are contained.
The PCR primer for the auxiliary screening of the cabbage self-compatible line is developed, has strong specificity and good stability, does not generate non-specific amplification at 55 ℃, and can very accurately detect whether the cabbage plant to be detected is recessive nuclear gene male sterility. In general, fertility of cabbage materials of different varieties can be determined only by manual observation in flowering phase, and a target strain can be rapidly screened in seedling phase by adopting the primer BoS1F/R to carry out auxiliary screening of cabbage affinity. Therefore, the PCR marker disclosed by the invention is adopted to carry out breeding auxiliary selection on the cabbage, so that the breeding period is greatly shortened, and the breeding efficiency is improved.
The invention provides a method for auxiliary screening of a cabbage self-affinity line, which is simple and easy to operate, and can judge whether the plants to be detected are affinity or not by extracting the genomic DNA of the cabbages to be detected, performing PCR reaction by adopting a primer BoS1F/R and detecting the length of a characteristic strip of a PCR product through agarose gel electrophoresis.
In conclusion, the PCR marker and the primer provided by the invention are used for screening the self-compatibility of the cabbage, the operation is simple and easy to implement, the specificity is strong, the stability is good, the breeding period can be greatly shortened, and the breeding efficiency is improved.
Drawings
FIG. 1 shows the amplification of the primers BoS1F/R in different inbred cabbage lines, in which,
M:Ladder;
1 is cabbage self-compatibility material '87-534';
2-17 self-fertile materials are respectively 96-100%, 01-20%, 02-12%, American early cabbage, summer peak, Mingde, delicious early growing, Zhonggan 21%, Zhonggan 23%, Zhonggan 25%, Zhonggan 196%, Huifeng No. 4 and suburb;
FIG. 2 shows F obtained for the parental construction with primers BoS1F/R at 87-534 and 96-1001、F2Amplification in strains, wherein P1 is 87-534, P2 is 96-100, F1Hybrids of 87-534 and 96-100; 1-14 are moieties F2And (5) strain.
Detailed Description
The present invention is further illustrated below by reference to specific examples, which are intended to be illustrative only and not to be construed as limiting the scope of the invention.
Biological material
Cabbage material: 87-534, 96-100, 01-20, 02-12, American early cabbage, summer peak, Mingde, delicious early-growing, middle sweet 21, middle sweet 23, middle sweet 25, middle sweet 196, Huifeng No. 4, suburb and 87-534 are self-compatible materials of cabbage; 87-534 and 96-100 for parental construction of F1、F2100 strains.
All the above biological materials are stored in laboratories and can be released to the public for verification tests within twenty years from the filing date.
The experimental reagents which are not particularly described in the invention are all conventional reagents in the field, or are prepared by adopting conventional methods in the field, can be obtained commercially, and have the specification of laboratory pure grade.
Example 1. development of markers for cabbage self-affinity assisted screening and their specific primers:
carrying out whole genome re-sequencing on the cabbage self-compatible material '87-534' and the self-fertile material '96-100', searching SNP and Indel differential sites in the materials, searching and screening DNA sequences at the site http:// 10.122.68.27/cgi-bin/gbrowse/Oleracea/searching and screening DNA sequences at the site with the differential sites, and then designing primers at the site http:// bioinfo. ut. ee/primer 3-0.4.0/DNA sequences passing through the differential sites;
screening all designed primers by using parent DNA, and searching primers with differences;
then using the screened differential primer pair F of 87-534 and 96-1002Establishing a linkage marker in a genome DNA mixed pool of generation materials, finding and determining a candidate gene, finding a PCR marker BoS1 according to the difference of the sequence of the candidate gene in parents '87-534' and '96-100', and designing and screening the following primers according to the marker:
upstream primer BoS 1F: 5 '-AAACTTGTATCTGAAGAC-3'
Downstream primer BoS 1R: 5 '-TCTCGTTCTTGCTAGTCTTC-3'
Example 2 kit and preparation thereof
The primers obtained by screening and designing in the example 1 are artificially synthesized and directly loaded into a centrifuge tube, or are loaded into the centrifuge tube after being prepared into 10-100 times of working concentration, and then are loaded into a kit.
Reagent containers for carrying out PCR and/or electrophoresis reagents are loaded into the kit.
Example 3 verification of the accuracy of the PCR primer or the kit for screening cabbage affinity
Reagent: 2 × EasyTaqPCRSuperMix purchased from Beijing Quanyujin
Step 1, extracting genomic DNA
Genomic DNA of cabbage "87-534", "96-100", "01-20", "02-12", "American early cabbage", "summer", "Mingde", "delicious early growing", "Zhonggan 21", "Zhonggan 23", "Zhonggan 25", "Zhonggan 196" and "Huifeng No. 4" was extracted by the CTAB (cetyl triethyl ammonium Bromide) method.
The DNA extraction method is described in Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA, nucleic Acids Res 8: 4321-4325.
Step 2, identifying the amplification condition of the primer BoS1F/R in the cabbage material
To identify the amplification of the primers in cabbage, Huada Gene Corp was entrusted with the synthesis of the primer BoS 1F/R. And (3) carrying out PCR reaction by using the genome DNA obtained in the step (1) as a template according to the following reaction system and reaction program.
The PCR reaction system comprises: the total volume of PCR reaction was 10. mu.L, wherein the upstream and downstream primers, BoS1F, BoS1R, (10. mu. mol/L) were each 0.5. mu.L, 2 × EasyTaqPCRSuperMix 5. mu.L, template DNA (20 ng/. mu.L) 2. mu.L, ddH2O2μL。
The conditions of the PCR reaction are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 45s, 35 cycles; extending for 7min at 72 ℃; storing at 4 ℃.
And (3) detecting the amplification product by electrophoresis in agarose gel, carrying out constant pressure detection at 150V for 30min, and taking pictures by an ultraviolet light gel imager.
The results are shown in FIG. 1: when the primer BoS1F/R is used for amplification of cabbage self-bred line materials, no characteristic band is detected in all cabbage samples, only cabbage affinity materials 87-534 are subjected to non-specific amplification, and a 93bp band is generated (as shown in Seq ID No. 3).
Example 4 screening of F constructed by 87-534 and 96-100 PCR primers or kits of the invention1And F2Accuracy verification of self-compatibility in population
Reagent: 2 × EasyTaqPCRSuperMix purchased from Beijing Quanyujin
Step 1, extracting genomic DNA
F obtained by extracting Brassica oleracea 87-534 and 96-100 with CTAB (cetyl triethyl ammonium Bromide) method1And 100 strains F2Population genomic DNA.
The DNA extraction method is described in Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA, nucleic Acids Res 8: 4321-4325.
Step 2, identifying F constructed by primers BoS1F/R at 87-534 and 96-1001And F2Amplification events in a population
To identify the amplification of the primers in cabbage, Huada Gene Corp was entrusted with the synthesis of the primer BoS 1F/R. And (3) carrying out PCR reaction by using the genome DNA obtained in the step (1) as a template according to the following reaction system and reaction program.
The PCR reaction system comprises: the total volume of PCR reaction was 10. mu.L, wherein the upstream and downstream primers BoS01F, BoS01R (10. mu. mol/L) were each 0.5. mu.L, 2 × EasyTaqPCRSuperMix 5. mu.L, template DNA (20 ng/. mu.L) 2. mu.L, ddH2O2μL。
The conditions of the PCR reaction are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 45s, 35 cycles; extending for 7min at 72 ℃; storing at 4 ℃.
And (3) detecting the amplification product by electrophoresis in agarose gel, carrying out constant pressure detection at 150V for 30min, and taking pictures by an ultraviolet light gel imager.
Primer BoS1F/R was used for F1、F2Specific amplification of the strain, amplified band at F1The single plant is a heterozygous strip; at F2Population amplified bands appear in three types: the belt type is 87-534, 96-100, F1In agreement, the PCR bands of the partial individuals are shown in FIG. 2.
And (3) field investigation: according to F2The affinity index of the individual plants of the group is investigated in the field, and the result shows that the affinity index is less than 1, which is taken as a self-incompatibility standard, and 24 compatible individual plants exist in the group.
And (3) PCR detection: for the same F2The pcr amplification of the individual plants in the population shows that the individual plants with the BoS1F/R amplification product banding pattern consistent with 87-534 are 25 plants, and the 25 plants comprise all 24 compatible individual plants found in field investigation.
The experimental data show that the primer BoS1F/R provided by the invention can accurately identify or screen the cabbage affinity material.
SEQUENCE LISTING
<110> vegetable and flower institute of Chinese academy of agricultural sciences
<120> PCR primer and kit for screening cabbage self-affinity material and application thereof
<130> P190120-SCH
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> PCR BoS 1-labeled upstream primer BoS1F for cabbage self-affinity screening
<400> 1
aaacttgtat ctgaagac 18
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> PCR BoS1 labeled downstream primer BoS1R for cabbage self-affinity screening
<400> 2
tctcgttctt gctagtcttc 20
<210> 3
<211> 91
<212> DNA
<213> cabbage
<220>
<223> characteristic band of PCR marker BoS1F/R for self-affinity screening
<400> 3
aaacttgtat ctgaagacaa catagtgttg acataggtcg aaagggccgg atgaaataga 60
atcaagacaa ggaagactag caagaacgag a 91

Claims (8)

1. The PCR primer for screening the self-affinity material of the cabbage (Brassica oleracea L.) has the nucleotide sequence as follows:
upstream primer BoS 1F: 5'-AAACTTGTATCTGAAGAC-3'
Downstream primer BoS 1R: 5'-TCTCGTTCTTGCTAGTCTTC-3'
The size of a characteristic band of the cabbage self-compatibility material amplified by the PCR primer is 91bp, and the nucleotide sequence of the characteristic band is shown as Seq ID No. 3.
2. A kit for screening cabbage self-affinity materials, comprising the PCR primer of claim 1.
3. The kit of claim 2, further comprising reagents required for performing PCR and/or electrophoresis.
4. A method for screening cabbage self-affinity materials, comprising performing the following steps using the PCR primer of claim 1 and/or the kit of claim 2 or 3:
(1) carrying out PCR on the genome DNA of the cabbage material to be detected by adopting the PCR primer;
(2) detecting the PCR product by gel electrophoresis;
(3) screening out a material with the size consistent with the characteristic strip size of the cabbage self-compatibility material from an electrophoresis detection result;
if the PCR product of the BoS1F/R has a characteristic strip of 91bp, the cabbage material to be detected has self-affinity characteristics;
if the PCR product of BoS1F/R has a characteristic strip of 80bp, the cabbage material to be detected does not have self-affinity characteristics.
5. The method according to claim 4, wherein the PCR reaction system for performing PCR on the genomic DNA of the cabbage material to be detected by using the PCR primers is as follows: template genomic DNA 0.2. mu.L/. mu.L, containing Mg2+0.1. mu.L/. mu.L of 10 XPCR buffer, 0.2mM of dNTPs, 0.5U/. mu.L of Taq DNA polymerase, 0.4. mu.M of forward and reverse primers, and the balance double distilled water.
6. The method of claim 4, wherein the PCR reaction conditions are: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 45s, 35 cycles; extending for 7min at 72 ℃; storing at 4 ℃.
7. The method of claim 4, wherein the gel electrophoresis detection is performed by using 8% polyacrylamide gel, performing electrophoresis separation at constant power of 160V for 70 minutes, and finally performing silver staining for color development.
8. The method for preparing a kit according to claim 2 or 3, wherein the PCR primer according to claim 1 is contained in a packaging box labeled for cabbage self-compatibility screening.
CN201910401144.0A 2019-03-13 2019-05-15 PCR primer and kit for screening cabbage self-affinity material and application of PCR primer and kit Active CN110512018B (en)

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CN111705157A (en) * 2020-07-24 2020-09-25 中国农业科学院蔬菜花卉研究所 PCR marker and primer for screening I-type S haplotypes of cabbage self-incompatible line
CN112980986B (en) * 2021-02-19 2022-04-15 中国农业科学院蔬菜花卉研究所 PCR primer group for identifying or screening cabbage hybrid lethal parent type 2 and application thereof
CN112980985B (en) * 2021-02-19 2022-04-12 中国农业科学院蔬菜花卉研究所 PCR primer group for identifying or screening cabbage hybrid lethal parent type 1 and application thereof

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CN103451283B (en) * 2013-08-20 2015-04-15 华中农业大学 Molecular detection method of Brassica napus self-incompatible S-locus haplotype
CN104293906B (en) * 2014-04-30 2017-06-23 南京农业大学 The SSR molecular marker of identification that Chinese cabbage selfing is not affine and its application

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